Nosocomial infections mainly are due to inefficient cleaning in association with the uncontrollable prescription of antimicrobials resulting in the emergence of multi-drug resistant pathogens in the hospital environment. Objectives:The study aims to evaluate the impact of the implementation of culture-guided antibiotic policy with strict infection control strategies on the occurrence of nosocomial infections and the resistance pattern ofthe isolated clinical and environmental pathogens. The study was done in 2 periods. Firstly, (August 2016 – April 2017), routine disinfection procedures and the applied antibiotic policy were evaluated. Secondly, according to the results a new antibiotic policy depending on the culture sensitivity results were implemented starting from June 2017 to February 2018 in association with strict infection control practices. As a result of this intervention, A change in the type of the isolated microorganisms was observed.Antibiotic resistance was decreased. Mortality rate was reduced from 14.1% to 9.5% of neonates with nosocomial infections, the number of the prescribed antibiotics didn’t exceed 4 antibiotics decreasing the overall cost for neonates’ therapy during their hospital stay. Each hospital should have its own antibiotic policy with the application of strict infection control strategies for the control of nosocomial infection.